Collagen and gelatin-based biomaterials have been successfully employed for a variety of tissue engineering applications (Rohanizadeh et al. J Mater Sci Mater Med 2008; 19: 1173-1182; Takemoto et al. Tissue Eng Part A 2008; 14: 1629-1638; Young et al. J Control Release 2005; 109: 256-274). Both of these macromolecules are characterized by excellent biocompatibility and low antigenicity (Cenni et al. J Biomater Sci Polym Ed 2000; 11: 685-699; Lee et al. Int J Pharm 2001; 221: 1-22; Waksman et al. J Immunol 1949; 63: 427-433); however, since gelatin is obtained by the hydrolysis of collagen, it has certain advantages over the latter: (a) it is readily available and easy to use; (b) offers options relative to molecular weight and bloom (i.e. control over physical properties); and (c) is more flexible towards chemical modification and more straightforward to manufacture. Moreover, from a biological standpoint, gelatin maintains cytocompatibility and cell adherence properties similar to collagen Engvall et al. Int J Cancer 1977; 20: 1-5; Kim et al. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2009; 108: e94-100).
Various methods have been reported for the crosslinking of these macromolecules for the purpose of delaying their biodegradation to prolong their in vivo residence (in tissue engineering applications) or tailoring their drug releasing capacity (when used as drug carriers). Numerous methods have been published for chemical or photochemical crosslinking of collagen or gelatin (Adhirajan et al. J Microencapsul 2007; 24: 647-659; Chang et al. Macromol Biosci 2007; 7: 500-507; Gagnieu et al. Biomed Mater Eng 2007; 17: 9-18; Kimura et al. J Biomater Sci Polym Ed 2010; 21: 463-476; Ma et al. J Biomed Mater Res A 2004; 71: 334-342; Vandelli et al. Int J Pharm 2001; 215: 175-184; Vandelli et al. J Control Release 2004; 96: 67-84). The majority of these procedures are targeted to reduce the susceptibility of these biomaterials to enzymatic degradation and to extend their in vivo residence time (Chang et al. supra 2007; Ma et al. supra 2004). Other crosslinking methods are typically employed to yield gelatin or collagen-based biomaterials suitable as slow release drug, protein or nucleic acid carriers (Kimura supra 2010; Vandelli supra 2004; Kommareddy et al. Nanomedicine 2007; 3: 32-42; Sehgal et al. Expert Opin Drug Deliv 2009; 6: 687-695; Sutter et al. J Control Release 2007; 119: 301-312). A widely used crosslinking agent class for collagen and gelatin as well as other tissue engineering-compatible systems is the carbodiimides (Adhirajan supra 2007; Olde Damink et al. Biomaterials 1996; 17: 765-773; Pieper et al. Biomaterials 2000; 21: 581-593; Cornwell et al. Clin Podiatr Med Surg 2009; 26: 507-523). These molecules are known as zero-length crosslinkers and act by mediating the formation of amide bonds between carboxyl and primary amine functionalities present on the species to be crosslinked. In addition, carbodiimides are less cytotoxic compared to other common crosslinking agent (e.g. glutaraldehyde) (Lai et al. J Mater Sci Mater Med 2010; 21: 1899-1911). Glutaraldehylde is used as a crosslinker in Cultispher™ beads. Burg U.S. Pat. No. 6,991,652 describes tissue engineering composites containing three-dimensional support constructs for cells that may be delivered to a subject.
Regenerative medicine technologies provide next-generation therapeutic options for chronic kidney disease (CKD). Presnell et al. WO/2010/056328 and Ilagan et al. PCT/US2011/036347 describe isolated bioactive renal cells, including tubular and erythropoietin (EPO)-producing kidney cell populations, and methods of isolating and culturing the same, as well as methods of treating a subject in need with the cell populations.
There is a need for therapeutic formulations that are suitable for delivery of active agents, such as for example, bioactive cells in tissue engineering and regenerative medicine applications, to subjects in need.
The kidney is a complex organ that performs many functions to keep the blood clean and chemically balanced. In addition to removing wastes, the kidneys release three important hormones:
erythropoietin, or EPO, which stimulates the bone marrow to make red blood cells
renin, which regulates blood pressure; and
calcitriol, the active form of vitamin D, which helps maintain calcium for bones and for normal chemical balance in the body.
To perform these functions the kidney comprises numerous different cell types. However, not all renal cells are required for a regenerative response and identifying the combination of cells useful in eliciting a regenerative response has been the subject of investigation. Thus, there remains a need for methods that identify a heterogeneous renal cell population, i.e. bioactive cells, that finds use in the therapeutic formulations disclosed herein.